Synthesis and activity of novel 5-substituted pyrrolo[2,3-d]pyrimidine analogues as pp60c-Src tyrosine kinase inhibitors

Article abstract of DOI:10.1002/ardp.200700141

Therapy with receptor tyrosine kinase inhibitors provides an improved treatment option in a number of diseases such as cancer, myocardial infection, osteoporosis, stroke, and neurodegeneration. We have designed, synthesized, and evaluated a series of nove

Full text of DOI:10.1002/ardp.200700141

Arch. Pharm. Chem. Life Sci. 2008, 341, 113–120
S. ꢀlgen et al.
113
Full Paper
Synthesis and Activity of Novel 5-Substituted Pyrrolo[2,3-
d]pyrimidine Analogues as pp60c-Src Tyrosine Kinase Inhibitors
Sꢀreyya ꢁlgen1, Yasemin G Isgꢂr1, and Tꢀlay ꢃoban2
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ankara, Tandogan-Ankara,
Turkey
2 Department of Toxicology, Faculty of Pharmacy, University of Ankara, Tandogan-Ankara, Turkey
Therapy with receptor tyrosine kinase inhibitors provides an improved treatment option in a
number of diseases such as cancer, myocardial infection, osteoporosis, stroke, and neurodegen-
eration. We have designed, synthesized, and evaluated a series of novel 2-amino-5-[(benzyl)imi-
no]methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidine-4-one 7a and 2-amino-5-[(substituted-benzyl)i-
mino]methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidine-4-one 7b-e derivatives as potential tyrosine
kinase inhibitors. These compounds were synthesized by condensation reaction using 2-trityla-
mino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbaldehyde 5 and appropriate benzyl-
amines followed by detritylation. Compounds were evaluated for their inhibitory activity
toward tyrosine phosphorylation for the pp60c-Src tyrosine kinase. Compounds 7a, 7d, and 7e
demonstrated potent inhibitory activities against pp60c-Src tyrosine kinase with IC50 values of
13.9, 34.5, and 78.4 lM, respectively. Dihalogenated compounds 7d and 7e have 3 to 7-times
lower IC50 values than that of the parent compound 7a.
Keywords: Cancer / Pyrrolopyrimidine derivatives / Schiff bases / Tyrosine kinase pp60c-Src
Received: July 3, 2007; accepted: October 8, 2007
/
DOI 10.1002/ardp.200700141
ily kinase activation, the catalytic activity of SFKs in nor-
mal mammalian cells is suppressed mainly by two inhib-
itors called C-terminal Src kinase (CSK) and CSK-homolo-
gous kinase (CHK), which inactivate SFKs by phosphory-
lating a consensus tyrosine near the C-terminus of SFKs.
Src-modulated intracellular signal transduction through
multiple pathways are heavily implicated in a number of
diseases such as cancer, diabetes, rheumatoid arthritis,
autoimmune diseases, stroke, myocardial infarction,
osteoporosis, and numerous diseases of the eye [4]. More-
over, novel roles of various Src family members in the
development of human leukemia and pancreatic adeno-
carcinomas have been recently reported [5]. New insights
into downstream signaling mechanisms, including the
activation of STAT3, PDK1, and Akt, further corroborate
the importance of Src family kinases in tumorigenesis
and chemoresistance [6]. All of these taken together
explain how Src activation affects tumor progression
through activation of several signaling molecules that
are known to contribute to tumor-cell survival and
increased metastatic potential. The normal cellular
Introduction
Protein tyrosine kinases (PTKs) are enzymes that selec-
tively phosphorylate tyrosine residues in different sub-
strates and have been shown to be extensively involved
in the formation and maintenance of cancer [1]. The Src
family kinases (SFKs) are the largest family of non-recep-
tor protein tyrosine kinases, which are responsible for
signal transduction during many cellular activities,
including differentiation, adhesion, and migration [2].
The Src family of tyrosine kinases comprise the highly
homologous proteins Blk, Brk, Fgr, Frc, Fyn, Hck, Lck,
Lyn, Src, Srm, and Yes [3]. To prevent constitutive Src fam-
Correspondence: Sꢁreyya ꢀlgen, Associate Professor, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ankara,
Tandogan, Ankara 06100, Turkey.
E-mail: olgen@pharmacy.ankara.edu.tr
Fax: +90 312 213-1081
Abbreviations: Protein tyrosine kinases (PTKs); Src family kinases
(SFKs)
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S. ꢀlgen et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 113–120
homologue (c-src) of the rous sarcoma virus oncogene (v-
src), the first molecularly defined proto-oncogene, enco-
des the pp60c-Src PTK (2ptk) [7]. The pp60c-Src is a non-recep-
tor tyrosine kinase that is characterized by a N-terminal
unique domain followed by two Src homology domains
SH3 and SH2, a kinase domain, and a short C-terminal
regulatory peptide segment. This protein is involved in
oncogenic signal transduction by the receptor tyrosine
kinases EGFR / HER1, HER2, and PDGFR [8]. The pp60c-Src
has been implicated in the development of leukemia,
breast, and colon cancer [9]. In addition, the inhibitors of
pp60c-Src tyrosine kinase particularly have been identified
as potential therapeutics for osteoporosis [10]. In the few
past years, much progress has been made about different
kinase inhibitors with utility in oncology; they often lack
selectivity or show weak cellular potency.
For the pp60c-Src inhibitor discovery and development,
several approaches have been adopted to inhibit or
decrease kinase activity. In general, inhibitors of kinase
activity fall into two broad classes: (i) inhibitors of the
tyrosine kinase activity (adenosine triphosphate [ATP]
binding domain-mediated) and (ii) inhibitors of protein–
protein (SH2-, SH3-, or substrate binding domain-medi-
ated) interactions. Inhibiting the activity of tyrosine kin-
ases by low molecular weight compounds (small mole-
cule inhibitors) that are capable of interfering with
either ligand binding (in the case of receptor tyrosine
kinases) [11] or with protein substrate (in case of non-
receptor tyrosine kinase) has proved to be difficult [12].
Therefore, the ATP competitive inhibitors appear to be
the target of choice [13] to discover and develop novel
inhibitors of Src family kinases. The ATP binding site,
though evolutionarily conserved, can be selectively tar-
geted by taking advantage of the minor difference in the
kinase domain. The minor difference leads to changes in
hydrogen bonding and hydrophobic interactions result-
ing in differences of affinity [14]. Compounds that are
found to be ATP mimics possess at least two aromatic
rings.
Figure 1. The structures of PP1, PP2, and LY231514 as TKIs
inhibitors.
yl-amine] and PP2[1-tert-butyl-3-p-chloro-1H-pyrazolo [3,4-
d]pyrimidine-4-yl-amine], which were found as selective
inhibitors of Src family member pp60c-Src (Fig. 1) [23].
Although these compounds inhibit SFK with IC50 in nano-
molar range with high selectivity for SFKs, usually PP1
and PP2 have been used to delineate SFK-dependent sig-
nal transduction pathways in vitro and in vivo. This is due
to their potency to inhibit “off-target” kinases in vitro at
sufficient concentrations to achieve complete SFK inhibi-
tion. More recently, several pyrrolopyrimidine com-
pounds (7-pyrrolidinyl- and 7-piperidinyl-5-pyrrolo[2,3-d]-
pyrimidines and 7-alkyl- and 7-cycloalkyl-5-aryl-pyr-
rolo[2,3-d]pyrimidines) were reported as potent tyrosine
kinase pp60c-Src inhibitors, which are useful in the treat-
ment of osteoporosis [24, 25]. 2-Amino-4-oxo-pyrrolo[2,3-
d]pyrimidine (LY231514, pemetrexed) (Fig. 1) is also a pyr-
rolopyrimidine compound, which was reported as a
strong anti-cancer agent [26]. Pyrrolopyrimidine and
furopyrimidine derivatives have been also developed as
inhibitors of folate metabolism. Folate metabolism has
long been recognized as an effective target for chemo-
therapy because of its crucial role in the biosynthesis of
nucleic acid precursors [17]. Inhibitors of folate-depend-
ent enzymes have been found as clinically useful antitu-
mor, antimicrobial, and antiprotozoal agents.
Recently, Src has emerged as an attractive candidate
molecule for targeted therapies, with development of
several small molecule inhibitors of Src family kinases
that may be of use in targeting tumor growth and meta-
stases. With an emphasis on combination therapies with
standard chemotherapeutic agents [27], Src kinase family
enzymes, and especially the pp60c-Src inhibition, may
exhibit the dual function by virtue of increasing the sen-
sitivity of tumors to established chemotherapeutic
agents and improve disease prognosis by preventing
tumors to metastasize.
The most potent and selective compounds with cellu-
lar activity belong to the related classes of heterocyclic
ATP analogs, namely pyrazolo-, pyrido- and pyrrolopyri-
midines. Among these classes of inhibitors, anilinoquina-
zolines [15], pyridopyrimidines [16], pyrrolopyrimidines
[17–20], and pyrazolopyrimidines [21] have been identi-
fied and exhibited promising in-vitro and in-vivo potency
toward several kinases. Several natural and synthetic pyr-
azolopyrimidine and pyrrolopyrimidines compounds
have been studied as selective SFK inhibitors [22]. Toward
the development of more effective SFK inhibitors, it is
important to draw attention to the pyrazolopyrimidines
PP1[1-tert-butyl-3-p-tolyl-1H-pyrazolo[3,4-d]pyrimidine-4-
Tyrosine kinase pp60c-Src has been, and still is, one of
the most-studied cellular protein tyrosine kinases. Sev-
eral SFK inhibitors, both of natural and synthetic origin,
have been reported with improved selectivity and
potency, and some of them have reached preclinical and
clinical investigation, yet none of them has reached the
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Arch. Pharm. Chem. Life Sci. 2008, 341, 113–120
5-Substituted Pyrrolopyrimidines as pp60c-Src Inhibitors
115
Scheme 1. The synthesis of 5-substituted pyrrolo[2,3-d]pyrimidine derivatives.
1
market either for osteoporosis or for cancer by targeting at 1008C. H-NMR indicated the regiospecific formation
tumor growth, cell adhesion, and mobility. Herein, we of the PreQ0 with no detectable formation of the corre-
1
report the studies on new promising compounds, 2- sponding 6-cyano-furo[2,3-d]pyrimidine 3. The H-NMR
amino-5-[(benzyl)imino]methyl-3,7-dihydro-4H-pyrrolo-
spectrum showed the characteristic downfield lactam
[2,3-d]pyrimidine-4-one 7a and 2-amino-5-[(substituted- NH signal at 11.96 ppm and the pyrrole NH signal at
benzyl)imino]methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyri-
midine-4-one 7b–e.
10.67 ppm. In addition, the presence of only one amino
group at 6.36 ppm and the absence of the pyrimidine 5-
proton between 5 and 6 ppm confirmed the structure of
the desired intermediate 2. Although a report on acetyl
protection of PreQ0, and the trityl protection of inter-
mediate compound 5 exists [29, 30], trityl protection was
Results and discussion
In this study, a practical synthesis of 2-amino-5-[(benzyl)i- chosen as the most suitable one to exploit a condensation
mino]methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidine-4- process leading to fully acid-labile compounds. The pres-
one 7a and 2-amino-5-[(substituted-benzyl)imino]methyl- ence of a trityl group in the resulting product provided
3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidine-4-one 7b–e us with the most soluble intermediate in a good yield. In
derivatives has been developed (Scheme 1). We required a addition to improving the solubility of the resulting
convenient method for the preparation of 2-tritylamino- intermediate compound, the trityl protection of the 2-
4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbalde- amino group has several other important advantages
hyde 5 as an intermediate. Information regarding the such as, the protection can easily be removed by
synthesis of compound 5, which is the key intermediate CF3COOH at room temperature under mild conditions
throughout the whole synthesis procedure, and also its [31], and the tritylated intermediate compound facili-
Schiff' base derivatives, is not available in the literature. tates the following reaction step in a reasonably good
Here, we describe the synthesis method for compound 5 yield. Whereas no yield was obtained with the unpro-
and its Schiff' bases
tected intermediate compound.
A previously reported procedure was used for the syn-
Although several methods are available for reducing a
thesis of 2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyri- cyano group to aldehyde [32, 33], in this study, the cyano
midine-5-carbonitrile (PreQ0) 2 [18, 28]. In the reaction of group was converted to aldehyde with DIBAL reduction
methyl formate with chloroacetonitrile in toluene, the with some modifications compared to the method
use of NaOCH3 afforded the chloroaldehyde 1 in quantita- reported in the literature [34]. In brief, among the tested
tive yield. Compound 1 was cyclo-condensed with 2,4-dia- conditions, the best one was the reaction carried out in
mino-6-hydroxypyrimidine and sodium acetate in water methylene chloride at 08C; it resulted in the intermediate
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Arch. Pharm. Chem. Life Sci. 2008, 341, 113–120
Table 1. Physicochemical properties and spectral data of 5-substituted-pyrrolo[2,3-d]pyrimidine derivatives.
Comp. Isomers Mp.
Yielda)
(%)
Molecular
Formula
IR/kmax
(cm– 1
Mass
1H-NMRb) DMSO-d6
(ratio)
(8C)
>300
65
)
2
4
36
79
C7H5N5O
6.36 (s, 2H, NH2-2), 7.59 (s, 1H, H-6),
10.67 (s, 1H, NH-7), 11.96 (s, 1H, NH-3)
7.20-7.33 (m, 15H, a.p.), 7.43 (s, 1H, NH-
2), 7.59 (s, 1H, H-6), 10.66 (s, 1H, NH-7),
11.96 (s, 1H, NH-3)
7.19-7.29 (m, 15H, a.p.), 7.39 (s, 1H, NH-
2), 7.55 (s, 1H, H-6), 9.99 (s, 1H, CHO),
10.64 (s, 1H, NH-7), 11.84 (s, 1H, NH-3)
4.02 (s, 2H, CH2-Ph), 4.87 (s, 2H, CH2-Ph),
7.21-7.40 (m, 22 H a.p., H-6, NH-2), 8.85
(s, 1H, CH=N), 8.73 (s, 1H, CH=N)
4.69 (s, 2H, CH2-Ph), 7.13-7.34 (m, 20 H,
a.p., NH-2), 7.57 (s, 1H, H-6), 8.59 (s, 1H,
CH=N)
3.94 (s, 2H, CH2-Ph), 4.65 (s, 2H, CH2-Ph),
7.19-7.40 (m, 20 H, a.p., NH-2), 7.46 (s,
1H, H-6), 8.58 (s, 1H, CH=N), 8.63 (s, 1H,
CH=N)
4.04 (s, 2H, CH2-Ph), 4.58 (s, 2H, CH2-Ph),
6.94 (s, 1H, NH-2), 7.17-7.39 (m, 18 H,
a.p.), 7.46 (s, 1H, H-6), 8.43 (s, 1H, CH=N),
8.66 (s, 1H, CH=N)
4.37 (s, 2H, CH2-Ph), 4.68 (s, 2H, CH2-Ph),
7.19-7.62 (m, 20 H, a.p., H-6, NH-2), 8.32
(s, 1H, CH=N), 8.63 (s, 1H, CH=N
Z: 4.68 (s, 2H, CH2-Ph), 6.53 (s, 2H, NH2-
2), 6.75 (s, 1H, H-6), 7.24-7.50 (m, 5 H
a.p.), 8.58 (s, 1H, CH=N), 10.40 (s, 1H,
NH-7), 11.86 (s, 1H, NH-3) E: 4.81 (s, 2H,
CH2-Ph), 6.59 (s, 2H, NH2-2), 7.38-7.50 (m,
6 H a.p., H-6), 8.70 (s, 1H, CH=N), 10.44
(s, 1H, NH-7), 11.88 (s, 1H, NH-3)
E: 5.10 (s, 2H, CH2-Ph), 6.70 (s, 2H, NH2-
2), 7.15-7.19 (m, 3 H-3‘,5‘, H-6), 7.33-7.37
(m, 2H, H-2‘,6'), 8.60 (s, 1H, CH=N), 10.60
(s, 1H, NH-7), 11.08 (s, 1H, NH-3)
4.99 (s, 2H, CH2-Ph), 5.10 (s, 2H, CH2-Ph),
6.92 (s, 2H, NH2-2), 7.08-7.51 (m, 5 H a.p.,
H-6), 8.14 (s, 1H, CH=N), 8.82 (s, 1H,
CH=N), 10.65 (s, 1H, NH-7), 11.99 (s, 1H,
NH-3)
5.07 (s, 2H, CH2-Ph), 7.05 (s, 2H, NH2-2),
7.19-7.67 (m, 4H a.p.), 8.88 (s, 1H, CH=N),
10.66 (s, 1H, NH-7), 11.62 (s, 1H, NH-3)
4.15 (s, 2H, CH2-Ph), 5.12 (s, 2H, CH2-Ph),
7.13 (s, 2H, NH2-2), 7.20-7.77 (m, 4H a.p.,
H-6), 8.24 (s, 1H, CH=N), 8.89 (s, 1H,
CH=N), 10.72 (s, 1H, NH-7), 11.74 (s, 1H,
NH-3)
C26H19N5O
2227 (CN)
3380
(indole NH)
1641 (CH=O)
3431
(indole NH)
1604 (CH=N)
3386
(indole NH)
1552 (CH=N)
3449
M++Na=440.2
(100%)
5
165
105
142
216
33
C26H20N4O
C33H27N5O
C33H26FN5O
C33H26ClN5O
M++1=421.19
(100%)
6a
6b
6c
E/Z
5/1
74.8
76.6
57.2
M++1=510.28
(100%)
E
M++1=528.30
(100%)
(indole NH)
1576 (CH=N)
3432
E/Z
2/1
M++1=544.25
(100%)
(indole NH)
6d
E/Z
20/1
172
268
61.4
C33H25F2N5O
1612, 1596
(CH=N) 3431
(indole NH)
M++1=546.35
(100%)
6e
7a
E/Z
1/1
80.5
66.7
C33H25Cl2N5O
C14H13N5O
1609 (CH=N)
3432
(indole NH)
1590 (CH=N)
3381
M++1=578.16
(100%)
E
E
>171
Decomp.
M++1=268.14
(100%)
(indole NH)
7b
7c
>199
Decomp.
60.4
C14H12FN5O
C14H12ClN5O
1562 (CH=N)
3446
(indole NH)
M++1=286.14
(100%)
E/Z
1/1
>200.6 37.4
Decomp.
1590 (CH=N)
3431
(indole NH)
M++1=302.17
(100%)
7d
7e
E
197
65.5
43.9
C14H11F2N5O
C14H11Cl2N5O
1596 (CH=N)
3468
(indole NH)
1555 (CH=N)
3493
M++1=304.08
(100%)
E/Z
1/1
>163
Decomp.
M++1=336.16
(100%)
(indole NH)
a)
Yield is given as Z/E mixture.
The 1H-NMR data of Z and E isomers are given together in mixture samples; a. p. = aromatic protons
b)
5 [35]. Many compounds and nearly all important func- l)imino]methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidine-
tional groups have been tested as adducts for highly reac- 4-one 7a and 2-amino-5-[(substituted-benzyl)imino]-
tive alkylaluminium hydrides [36]. Their popularity has methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidine-4-one
risen considerably due to several reasons, such as, diisobu- 7b–e compounds were obtained by the reaction of alde-
tylaluminium hydride has proved to be safe and easy-to- hyde 5, with appropriate amines (Scheme 1) followed by
handle in toluene or hexane. The target 2-amino-5-[(benzy- the removal of trityl protection [37]. All resulted products
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Arch. Pharm. Chem. Life Sci. 2008, 341, 113–120
5-Substituted Pyrrolopyrimidines as pp60c-Src Inhibitors
117
Table 2. Inhibitory concentrations (IC50; lM) of 5-substituted pyr-
rolo[2,3-d]pyrimidine derivatives against pp60c-Src
.
Compound
Isomers
IC50 (lM) X l S.D.
7a
7b
7c
7d
7e
E
E
E/Z
E
E/Z
13.98 l 4.06
> 200
174.30 l 86.5
34.50 l 3.75
78.36 l 4.81
were purified by recrystallization from the ethanol. Puri-
fication on a silica-gel column was performed before
recrystallization, only if it was found necessary. The struc-
ture of all synthesized compounds was confirmed by 1H-
NMR, IR, and MS spectral data as shown in Table 1. Both
protected and deprotected 5-substituted pyrrolo[2,3-
d]pyrimidine analogues 6a–e and 7a–e were obtained in
good yield. Under the standard conditions, compounds
were obtained as a mixture of Z/E-isomers. In general,
attempts to separate the Z/E mixtures were extremely tedi-
ous. In some cases, the repeated flash chromatographic
separations, followed by recrystallization, resulted in par-
tial separation of the pure E-isomer. The Z-isomer could
not be obtained either free or in good yield (a10%). In one
case, compound 7a was obtained in analytic amounts
only and characterized as Z-isomer. The data of the iso-
mers are reported in Table 1. Configuration of the sepa-
rated isomers was determined using NOE analysis. The E-
configured compounds have a NOE effect between the
proton at the imine (CH=N) and the proton(s) at the C-6
substitution of the 5-substituted pyrrolo[2,3-d]pyrimidine
compounds. These assignments were also confirmed by
1H-NMR spectral data, where the imine protons displayed
a slight down-field shift in the trans-(E) isomers than cis-(Z)
isomers. Their infrared spectra provided good evidence
for their structure, showing clearly both CH=N (1552–
1612 cm– 1) and indole NH peaks (3381–3493 cm– 1) in the
expected regions. The CH=N stretching values of Z isomers
were found higher then E isomers. For compound 2, the
CN stretching was detected at 2227 cm–1.
Figure 2. The dose-response curves for compound 7a with IC50
0.0139 mM, in the ELISA-based in-vitro pp60c-Src tyrosine kinase
assay with an initial concentration of 2 lM to a final concentra-
tion of 400 lM. The IC50 values obtained by the dose-response
curves for each compound (n = 3–4, two independent experi-
ments each in duplicates) were obtained by non-linear regres-
sion analysis using Bio Data Fit 1.02 and Origin 6.0.
tyrosine kinase activity with respect to the activity meas-
ured in the presence of vehicle alone (dimethyl sulfox-
ide). The dose response curves of the most active com-
pounds 7a and 7d were shown in Figs. 2 and 3, respec-
tively.
The most potent inhibition was observed with com-
pound 7a (IC50 = 13.9 lM), which has no substituents in
the benzyl ring. In addition, compounds 7d and 7e
showed good in-vitro efficacies at IC50 values of 34.5 and
78.4 lM, respectively. The benzyl-ring substitutions with
an electron-withdrawing fluorine and chlorine atoms at
the 2- and/or 4-positions result in a 3 to 20-fold decrease
for the inhibition of pp60c-Src tyrosine kinase. The better
inhibitory effect of dihalogenated over monohalogen-
ated compounds can be explained with the molecule's
3D conformation and flexibility to compete with ATP.
Here, we can hypothesize that the mono-halogen substi-
tution on the aromatic ring, possibly results in more flex-
ible compounds, which may not easily fit into the ATP-
binding site of Src, and hence, can not compete with ATP
in the medium. Introducing conformational constraints
into rigid dihalogenated compounds 7d and 7e generate
better interaction with the binding pocket and provide
more activity. In the process of binding, different con-
formers of the target and a given ligand may fit in one of
the most favorable energetic states. The dynamic nature
of both proteins and ligands can determine the interac-
tions and biological activity.
The biological response of the compounds was eval-
uated for their inhibitory activity towards tyrosine phos-
phorylation for the pp60c-Src tyrosine kinase [38]. Briefly,
the biological activity was monitored by the kinase-cata-
lyzed transfer of c-phosphate residue from ATP to immo-
bilized peptide substrates, and the results were expressed
as% inhibition of the c-Src activity in the presence of vary-
ing concentrations of compounds (dose-response curves)
7a–e. The inhibitory effect of compounds was reported
as IC50 of the compounds and is summarized in Table 2.
The IC50 values were defined as the concentration of a
compound required to achieve 50% inhibition of pp60c-Src
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S. ꢀlgen et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 113–120
for the activity loss. In the view of these findings, our
future goal should be oriented to investigate the effects
of new substituents at N5 and N7 positions.
In conclusion, noteworthy activity results of our new,
strategic 5-substituted pyrrolo[2,3-d]pyrimidine deriv-
atives, which exhibit potent inhibition of pp60c-Src tyro-
sine kinase, are reported here.
This work was partially supported by a grant from the Turkish
Scientific and Technical Research Institute (106S127 SBAG-HD-
141)
The authors have declared no conflict of interest.
Experimental
Figure 3. The dose-response curves for the compound 7d with
IC50 0.0345 mM, in the ELISA-based in-vitro pp60c-Src tyrosine
kinase assay with an initial concentration of 2 lM to a final con-
centration of 400 lM. The IC50 values obtained by the dose-
response curves for each compound (n = 3–4, two independent
experiments each in duplicates) were obtained by non-linear
regression analysis using Bio Data Fit 1.02 and Origin 6.0.
Chemistry
Melting points were measured with a capillary melting point
apparatus (Electrothermal 910, Essex, UK) and are uncorrected.
The Nuclear Magnetic Resonance (1H-NMR) spectra were
recorded on a Varian Mercury 400 NMR spectrometer for
400 MHz (Varian Inc., Palo Alto, CA, USA). The chemicals-shift
values were expressed in parts per million (ppm) relative to tetra-
methylsilane as an internal standard and signals are reported as
s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet). Mass
spectra were recorded on a Waters ZQ Micromass LC-MS spec-
trometer (Waters Corporation, Milford, MA, USA) Electrospray
Ionization (ESI) method. Infrared (IR) spectra were measured on
Jasco FT/IR-420 (Jasco, Tokyo, Japan). Elemental analysis was
taken on a Leco-932 CHNS-O analyzer (Leco, St. Joseph, MI, USA).
Element compositions are within l 0.4% of the calculated values.
Molecular Devices Spectra MAX 190 (from Molecular Devices
Corporation, Sunnyvale, CA, USA) was used to measure of
absorbance of the phosphorylation reaction. Analytical TLC was
carried out on Merck 0.2 mm pre-coated silica gel (60F 254) alumi-
nium sheets (Merck, Darmstadt, Germany), with visualization by
irradiation with a UV lamp. The flash-column chromatography
was accomplished on silica gel 60 (230–400 mesh, Merck).
Anhydrous magnesium sulphate, sodium sulphate, sodium
acetate, hexane, ethyl acetate, anhydrous toluene, trifluoroace-
tic acid, sodium dihydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, dimethylsulfoxide, sulfuric acid,
mercaptoethanol, celite (Merck, Darmstadt, Germany); deutero
dimethylsulfoxide, 2,6-diamino-6-hydroxy pyrimidine, sodium
methoxide, benzylamine, 4-chlorobenzylamine, 4-fluorobenzyl-
amine, 2,4-dichlorobenzylamine, 2,4-difluorobenzylamine,
methyl formate, dry pyridine, chloroacetonitrile, diisobutylalu-
minium bromide, trityl chloride (Acros Organics, Geel, Bel-
gium); methanol, hydrochloric acid, dichloromethane, potas-
sium hydroxide, sodium carbonate, anhydrous ethanol, etha-
nol, toluene, anhydrous toluene (Riedel-de Hꢀen, Sigma-Aldrich,
St. Louis, MO, USA) were purchased. Takara Universal Tyrosine
Assay Kit (from Takara-Bio Inc., Shiga, Japan) was used to test our
synthesized compounds. The contents of a kit: PTK substrate
immobilized microplate (8 wells612 wells), kinase reacting sol-
ution (11.0 mL), 40 mM ATP-2Na (0.55 mL), extraction buffer
(11.0 mL), PTK control (0.50 mL), anti-phospho-tyrosine (PY20-
HRP, for 5.5 mL/H2O), blocking solution (11.0 mL), HRP coloring
solution (TMBZ = tetra methyl benzidine, 12.0 mL).
In this context, considering that the geometric orienta-
tion of isomers can cause the diverse activity pattern, the
inhibitory potency of E- and Z-isomers may also be differ-
ent. Perhaps, the reason for the two-fold lower activity of
compound 7e compared to that of 7d may be due to the
presence of the E- and Z-isomers as a mixture. Since the
most active compound 7a has no substituent in the aro-
matic ring, this indicates that here, the electron-with-
drawing groups are not favorable for the biological activ-
ity. In the view of these results, it appeared necessary to
investigate more substitutions particularly more hydro-
philic substituent replacement to better understand the
structure-activity relationship. Taking into consideration
the receptor-ligand interactions on biological activity, it
can be concluded that hydrophobic interactions are non-
directional for our 5-substituted pyrrolo[2,3-d]pyrimidine
compounds. H-bonds are more directional and are
needed within the protein to enhance the c-Src inhibi-
tion.
The most active pyrrolo- and pyrazolopyrimidine com-
pounds have a N5-substituted phenyl ring and N7-alkyl- or
-heterocyclic substituents. It was assumed that N7-sub-
stituents of pyrrolopyrimidine occupies the pocket
which is usually utilized by the ribose moiety of ATP.
Both pyrrolo and pyrazolopyrimidine compounds bear-
ing substitute 5-phenyl ring were reported as favorable
selective c-Src inhibitors [24, 25]. The structural differen-
ces of our compounds at N5 and N7 positions compared to
active pyrrolopyrimidine compounds may be responsible
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Arch. Pharm. Chem. Life Sci. 2008, 341, 113–120
5-Substituted Pyrrolopyrimidines as pp60c-Src Inhibitors
119
2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-
5-carbonitrile 2
2-Amino-5-[(benzyl)imino]methyl-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidine-4-one 7a and 2-Amino-5-
[(substituted-benzyl)imino]methyl-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidine-4-one derivatives 7b–e
Compounds 6a–e were dissolved in anhydrous ethanol
(1 mmol, 10 mL) and CF3COOH (1 mmol, 10 mL, containing 15%
anhydrous dichloromethane) was added and stirred for 3 h at
room temperature. The solvent was removed in vacuo and 5%
NaHCO3 was added. The precipitate was collected, purified by
silica gel column chromatography (CH2Cl2/MeOH; 8 : 2) and crys-
tallized from ethanol.
Methyl formate (4.76 mL, 0.077 mol) was added to a stirred mix-
ture of 3.57 g (0.066 mol) NaOCH3 in anhydrous toluene at 08C.
Chloroacetonitrile (4.18 mL, 0.066 mol) was added dropwise
over 30 min at 08C and the reaction mixture was stirred at 08C
for 3 h. Water (100 mL) was added and extracted with toluene
(26100 mL). The aqueous phase was cooled at 08C and acidified
to pH 4 using 5 N HCl; then, it was extracted with 36100 mL
EtOAc. The organic phase was dried over Na2SO4. Concentrated
to dryness, it gave a crude compound, which was used without
further purification. Chloro(formyl)acetonitrile (3.98 g) 1 was
obtained as an oily compound. Sodium acetate (5.36 g,
0.065 mol) was dissolved in 118 mL of distilled water and 2,6-dia-
mino-6-hydroxy pyrimidine (4.12 g, 0.033 mol) was added. The
mixture was heated at 1008C and compound 1 in 62 mL of water
was added. The reaction mixture was refluxed for 5 h and it was
cooled to room temperature. The precipitate was collected by fil-
tration and washed with copious amounts of water and acetone.
The compound was dissolved in 30 mL of 6 N KOH, boiled with
charcoal, and then filtered over celite. The pH was adjusted to 6
at 08C with 30% HCl. The pure yellow solid (2.21 g, yield 58.7%)
was collected and dried in a vacuum oven at 508C for 24 h.
Inhibition of pp60c-Src tyrosine kinase
The ELISA-based in vitro tyrosine kinase assay, here the pp60c-Src
tyrosine kinase activity, was used to verify the inhibition of the
compounds synthesized in our laboratory. Briefly, the assay is
based on monitoring the phosphorylation of immobilized pep-
tide (substrate) by virtue of classical ELISA sandwich method,
where the phosphorylation of immobilized poly(Glu-Tyr) was
probed with HRP-conjugated anti-phosphotyrosine (PY20) anti-
body. In the assay, the inhibitory activities of compounds
against pp60c-Src tyrosine kinase were monitored as the dimin-
ished activity of kinase at 450 nm [38]. The kinase assay was per-
formed at 378C in a final assay volume of 50 lL. The concentra-
tions of the PTKs used to construct the calibration curve were as
follows: 1520, 760, 380, 190, 94.8, 47.4610– 7 units/lL for pp60c-
4-Oxo-2-tritylamino-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidine-5-carbonitrile 4
Compound 2 (1.05 g, 5.99 mmol) was suspended in dry pyridine
(65 mL) and trityl chloride (2.51 g, 8.99 mmol) was added to this
suspension. The reaction was warmed to 908C and stirred for
48 h. Water (3.2 mL) was added and the mixture was stirred for
20 min. The solvent was removed under reduced pressure and
the crude product was co-evaporated with toluene and ethanol,
providing a solid residue, which was purified by flash chroma-
tography (hexanes/EtOAc, 1 : 1).
Src
PTK. The kinase reactions were initiated by the addition of
40 nM ATP (10 lL) into each well, and the plate was subsequently
incubated at 378C for 30 min. After the completion of the reac-
tion, the remaining liquid was removed and the wells were
washed with PBS-Tween (0.05%, v/v) four times. The blocking sol-
ution (100 lL) was added to the wells and incubated at 378C for
30 min. After washing the plate with PBS-Tween, 50 lL of anti-
phosphotyrosine was added to the wells and incubated at 378C
for 30 min. Then, the liquid in the wells was decanted and the
remaining solution was removed by rinsing with PBS-Tween
four times. 100 lL of HRP coloring agents was added to each well
and incubated at 378C for 15 min. Reaction was terminated by
the addition of 100 lL 1 N sulfuric acid into each well. The
absorbance of the reaction mixture was measured at 450 nm
with a microplate reader.
4-Oxo-2-tritylamino-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidine-5-carbaldehyde 5
Compound 4 (0.59 g, 1.4 mmol) was dissolved in 14 mL anhy-
drous dichloromethane. DIBAL-H (1 M in dichloromethane,
4.24 mL, 4.24 mmol) was added at 08C over period of 30 min.
After 3 h, 4 mL of MeOH was added slowly at 08C and stirred for
30 min. The precipitate was filtered off and washed with MeOH.
The organic phase was dried over anhydrous MgSO4 and evapo-
rated to dryness to yield the crude compound 5, which was puri-
fied by flash chromatography (CH2Cl2/MeOH, 9.8 : 0.2).
The pp60c-Src tyrosine kinase activity is measured as the differ-
ence between the total activity of no vehicle (DMSO) and the
activity of enzyme in the presence of DMSO. The IC50 value is
defined as the concentration of a compound required to achieve
50% inhibition of pp60c-Src tyrosine kinase activity with respect
to the activity measured in the presence of vehicle alone
(dimethyl sulfoxide). The dose-response curves of the com-
pounds were obtained by using dilutions at final concentrations
of 400 to 2 lM in the ELISA-based in vitro protein kinase assay
described above and IC50 values were determined by non-linear
regression analysis. The dose-response curves of the compounds
and non-linear regression analysis were performed using Origin
6.0, (for Windows, Microcal Software, Northampton, MA, USA)
and BioDataFit 1.02 (Chang Bioscience, Inc., Castro Valley, CA,
USA).
2-Tritylamino-5-[(benzyl)imino]methyl-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidine-4-one 6a and 2-Tritylamino-5-
[(substituted-benzyl)imino]methyl-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidine-4-one derivatives 6b–e
Compound 5 (1 eq.) was dissolved in ethanol (1 mmol, 2 mL). The
appropriate benzylamine (1.5 eq.) and Na2CO3 (1 eq.) in water
(1 mmol, 0.5 mL) were added at room temperature, respectively.
The reaction mixture was stirred at 50–608C for 2 h. The solvent
was removed in vacuo and the crude compound was purified by
silica gel chromatography (CH2Cl2/MeOH; 9.5 : 0.5).
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www.archpharm.com

120
S. ꢀlgen et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 113–120
[20] A. Gangjee, A. Vidwans, E. Elzein, J. J. Mc Guire, et al., J.
Med. Chem. 2001, 44, 1993–2003.
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